Oncometabolic role of mitochondrial sirtuins in glioma patients

Mitochondrial sirtuins have diverse role specifically in aging, metabolism and cancer. In cancer, these sirtuins play dichotomous role as tumor suppressor and promoter. Previous studies have reported the involvement of sirtuins in different cancers. However, till now no study has been published with respect to mitochondrial sirtuins and glioma risks. Present study was purposed to figure out the expression level of mitochondrial sirtuins (SIRT3, SIRT4, SIRT5) and related genes (GDH, OGG1-2α, SOD1, SOD2, HIF1α and PARP1) in 153 glioma tissue samples and 200 brain tissue samples from epilepsy patients (taken as controls). To understand the role of selected situins in gliomagenesis, DNA damage was measured using the comet assay and oncometabolic role (oxidative stress level, ATP level and NAD level) was measured using the ELISA and quantitative PCR. Results analysis showed significant down-regulation of SIRT4 (p = 0.0337), SIRT5 (p<0.0001), GDH (p = 0.0305), OGG1-2α (p = 0.0001), SOD1 (p<0.0001) and SOD2 (p<0.0001) in glioma patients compared to controls. In case of SIRT3 (p = 0.0322), HIF1α (p = 0.0385) and PARP1 (p = 0.0203), significant up-regulation was observed. ROC curve analysis and cox regression analysis showed the good diagnostic and prognostic value of mitochondrial sirtuins in glioma patients. Oncometabolic rate assessment analysis showed significant increased ATP level (p<0.0001), NAD+ level [(NMNAT1 (p<0.0001), NMNAT3 (p<0.0001) and NAMPT (p<0.04)] and glutathione level (p<0.0001) in glioma patients compared to controls. Significant increased level of damage ((p<0.04) and decrease level of antioxidant enzymes include superoxide dismutase (SOD, p<0.0001), catalase (CAT, p<0.0001) and glutathione peroxidase (GPx, p<0.0001) was observed in patients compared to controls. Present study data suggest that variation in expression pattern of mitochondrial sirtuins and increased metabolic rate may have diagnostic and prognostic significance in glioma patients.


Introduction
Gliomas are among the most common type of CNS tumors and epidemiological studies have stated that gliomas are 40% of all CNS and brain tumors [1][2][3][4]. On the basis of molecular and

Expression analysis
Primers specific for the selected genes SIRT3, SIRT4, SIRT5, SOD1, SOD2, OGG1-2α, PARP1, GDH, HIF1α and β-actin was designed using the integrated DNA technology (IDT) as previously described by Ul Haq et al., [16]. Expression levels of above mentioned genes were measured using the Quantitative real time polymerase chain reaction (q-RT PCR).
Step one Plus Thermal cycler (Applied Biosystems) was used for this analysis. Each reaction constituted of 4 μl SYBR green, 2 μl of primers, 1 μl cDNA and 3 μl of PCR water. q-RT PCR conditions included, 10 min of initial denaturation, 1 min of annealing for 40 cycles. Cyclic threshold (CT) values were taken for each sample and 2 -ΔΔCt method was used to calculate relative expression.

Validation by NCBI GEO omnibus dataset
Involvement of these genes was assessed in genome wide expression in one dataset. The dataset containing glioma samples along with control samples was selected. The publicly available data to compare with our PCR results was obtained from NCBI Gene Expression Omnibus (GEO) database, (https://www.ncbi.nlm.nih.gov/geo/). Experiment type was Expression profiling by array with Accession number GSE:4290, Platform GPL570 (HG-U133_plus_2) Affymetrix Human Genome U133 Plus 2.0 Array [16].

Comet assay
Single cell gel electrophoresis was carried out for the estimation of DNA damage in glioma patients and healthy controls. Procedure adopted was carried out from Akram et al., [34] with some modifications. Glioma tissue samples of different subtypes along with healthy control tissues were used. The tissues were homogenized in saline solution and single cell suspension was prepared which was proceeded for detection of overall DNA damage in the tissues. Glass slides used in the procedure were labelled according to different glioma types. Three layered procedure was used the samples were sandwiched in the layers of low melting point (LMP) agarose gel. Whole area under coverslip was visualized and scored by fluorescent microscope. After running, mean %DNA damage, mean OTM (olive tail moment) and mean tail length were obtained and further processed for analysis. Comets were analyzed in Metafer 4 (MetaSystems).

Metabolic assays
ATP level determination. Glioma tissue sample and controls taken from patients were treated with saline solution to prepare tissue lysate. Three aliquots were prepared, one aliquot was used for the measurement of ATP level determination, as described by Afzal et al., [33]. Second aliquot was used for oxidative stress measurement and third for DNA damage assessment. For ATP level measurement, cell lysate of glioma and healthy controls were dispensed in micro titer plate and Cell titer glo (CTG) buffer was then added in the micro titer plate and Luminescence was recorded and analyzed. Oxidative stress determination. Glutathione level was measured from isolated protein lysate using Glutathione Assay Kit II (Calbiochem) according to manufacturer protocol provided with kit, as described by Afzal et al., [33].
Oxidative stress was measured by the cell lysate of the glioma samples and healthy controls. Oxidative stress was evaluated by quantitative analysis of three antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). Enzyme analysis was carried out using human specific ELISA kits purchased from Abcam (UK).
Absorbency of enzymes was checked in microplate reader (Platos R496, AMP Diagnostics), and sample concentrations were read from calibration curve. NAD level determination. NAD level was also measured by designing the primers for three important enzymes regulating NAD levels, they are NAMPT, NMNAT1 and NMNAT3. The q-RT-PCR procedure and program designing was same as stated above for expression analysis.
Data analysis. Statistical tools from SPSS and GraphPad Prism were used to analyze the data. SIRT3, SIRT4, SIRT5, SOD1, SOD2, GDH, mt OGG1-2α, PARP1 and HIF1a expression values were compared using student t-test, DNA damage values were compared using student t-test and one sample t-test while NAD+ and ATP values were also compared using student ttest. Gene to gene and gene to different histopathological parameters were compared by applying spearman correlation and diagnostic value was checked by ROC analysis. Survival analysis was performed by Kaplan Maier analysis.
Validation by NCBI GEO Omnibus dataset. Relative expression of our selected genes was validated from dataset GSE4290, titled: Expression data of glioma from Henry Ford hospital, at first data was normalized and analyzed in GraphPad Prism. Data set consisted of 157 glioma samples and 23 Epilepsy samples which were used as controls. 157 tumor samples include 26 astrocytoma, 50 oligodendrogliomas and 81 glioblastomas. Data analysis showed that SIRT4 (p<0.0001), SIRT5 (p = 0.0076), OGG1-2α (p<0.0001), SOD1 (p<0.0001) and SOD2 (p<0.0001) showed significant downregulation as shown in Fig 1B and 1D. while GDH (p = 0.6776) showed non significantly downregulated results (Fig 1D). In case of SIRT3 (p = 0.0142) and HIF1α (p = 0.0385) significant upregulation was observed (Fig 1B and 1D) while non-significant upregulated expression was observed in PARP1 (p = 0.203) in glioma samples as compared to healthy samples. Results are shown in Fig 1B and 1D.

Comet assay
For DNA damage assessment, comet assay was performed for glioma samples and uninvolved healthy controls. In this assay number of comets were assessed in glioma sections Fig 3A2  compared to the healthy controls Fig 3A1. Data analysis showed significantly higher number of comets in tumor samples (p = 0.04) compared to the controls (Fig 3B).
In the assay different parameters were also calculated which are %DNA in tail and it showed significant higher %DNA in tail in glioma patients compared to controls (p = 0.0005), HGG compared to LGG (p = 0.0374) and smokers in comparison with non-smokers (p = 0.0320) as shown in Fig 3C and 3F. In case of olive tail moment, it was observed significantly higher in

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glioma tissue samples compared to controls (p = 0.008), smokers compared to non-smokers (p = 0.0266) and non-significantly higher in case of HGG as compared to LGG (p = 0.1582) as shown in Fig 3D and 3F. Tail length was also assessed in patients and controls, tail length was observed higher in glioma patients compared to controls (p = 0.0005), HGG compared to LGG (p = 0.0011) while non-significantly less in smokers as compared to non-smokers (p = 0.1605) as shown in Fig 3E and 3F, Table 2.

Metabolic assays
ATP level was determined in glioma samples compared to the healthy controls. Analysis showed that ATP level was found significantly higher (p<0.03) in glioma compared to the control samples as shown in Fig 4A. ATP levels were also found to be significantly higher in HGG as compared to LGG and non-significantly low in smokers as compared to non-smokers, as shown in Fig 4B. Glutathione level was also assessed, and it was found to be significantly higher in glioma patients compared to controls (p<0.0001) and in HGG as compared to LGG (p<0.001), as shown in Fig 4C and 4D. While in case of smokers, glutathione level was found to be non-significantly higher as compared to non-smokers Fig 4D. NAD+ level was also determined by measuring the relative expression levels of NMNAT1, NMNAT3 and NAMPT. NMNAT1 was found significantly upregulated in gliomas (p<0.001) compared to controls and in HGG (p<0.001) compared to LGG patients. While in case of smokers, NMNAT1 was found non significantly upregulated compared to non-smokers as

Measurement of oxidative stress
Oxidative stress was evaluated in study cohort by measuring the three main antioxidant enzymes which includes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) as shown in

Spearman correlation
Gene to gene correlation was performed by spearman mentioned in Table 3. Negative correlation was observed between SIRT3 and SIRT4 (r = -0.

ROC analysis
To assess the diagnostic value of these selected genes, ROC curve analysis was performed as shown in

Discussion
Gliomas are most common intra-axial brain tumors originated from glial cells [5]. Risk factors for glioma include ionizing radiations, diet, exposure to pesticides, viruses, and genetic factors [35]. Genetic susceptibility also plays an important role in carcinogenesis and different pathways work to maintain genetic mechanisms. These pathways include mitochondrial DNA encoded genes as well as nuclear-encoded mitochondrial proteins [36]. Sirtuin family is among nuclear-encoded mitochondrial proteins, they are family of orthologues of yeast silent information regulator 3 (SIRT3), 4 (SIRT4) and 5 (SIRT5). Many studies have been proposed to discuss the role of sirtuins in cancers but still no study has been published to show their role with gliomas. The aim of present study was to elucidate the role of expression deregulations of mitochondrial sirtuin genes and co-expression with their associated proteins (SOD1, SOD2, OGG1-2α, PARP1, GDH and HIF1α) in glioma. Diagnostic/prognostic value of these genes was also analyzed in the glioma samples to check the role of these genes in glioma diagnosis. Metabolic assay and oxidative stress assay were also performed to assess the nonmetabolic role of selected genes in glioma tumoriogenesis.
SIRT3 is NAD+ dependent mitochondrial deacetylase, it is usually present in nucleus but in stress condition it is transported to mitochondria where it gets trimmed and activates. It regulates many important biological functions like metabolic control, gene expression, aging and many diseases like cancer [37]. Many studies have reported that metabolic programs are linked to tumorigenesis, as SIRT3 is an important mediator of mitochondrial metabolic pathways so

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SIRT4 is also found working in mitochondria, it controls the working of glutamate dehydrogenase (GDH), which ultimately controls the glutamate and glutamine activity by ADPribosylation. SIRT4 is found working as suppressor of glutamine under stressful condition, like oxidative stress and stabilizes the genomic stability [44]. Results from present study showed the significant downregulation of SIRT4 in glioma patients compared to healthy controls depicting its role as a tumor suppressor. Previous studies have also showed the downregulation of SIRT4 in gastric [45], thyroid [46] and breast cancer [47]. Mahjabeen & Kayani (2017) have reported that reason behind the downregulation of SIRT4 gene can be the variation in coding/ noncoding region of said gene, loss of functionality, abnormal metabolic rate, and increased carcinogenesis [17].
SIRT5 is another member of mitochondrial sirtuins, it is also found working in deacetylase activity. SIRT5 binds to glutaminase, which is first enzyme for glutamine metabolism [48]. In this study SIRT5 is found to be downregulated in glioma patients as compared to the healthy controls depicting its role as a tumor suppressor. In previous studies SIRT5 has been found downregulated in different cancers like liver cancer [48] gastric cancer [49] and hepatocellular carcinoma [50]. Previous studies have reported the upregulation of SIRT5 gene in breast [51], ovarian [20] and liver cancer [48]. Reason behind the downregulated and upregulated expression of SIRT5 gene is not clear yet. However, previous study has reported that high SNPs frequency in coding/noncoding region of SIRT5 gene resulted in deregulation of said genes, which ultimately results in increased reactive oxygen species, oxidative stress and carcinogenesis [52,53]. SOD1 is mitochondrial protein and act as an antioxidant enzyme. It is involved in formation of hydrogen peroxide to superoxide. SOD1 is a cytosolic protein but is also localized in inter-membrane spaces of mitochondria [54,55]. In present study SOD1 expression was found downregulated in gliomas compared to healthy control tissues which are in line with the previously reported findings. In previous studies, SOD1 has found to be overexpressed in numerous cancers like NSCLC [22], leukemia [56] and breast cancer [57], the overexpression is linked to poor survival of cancer cell [22]. Although SOD1 is highly expressed and act as oncogene in many cancers but in pancreatic cancer cells SOD1 has differentially expressed. It indicated that it is tissue-specific and play different and opposite roles in different types of cancers. High SOD1expression promotes tumor cell proliferation, it can be, by reduced antioxidant protein activity leading to increased intracellular H2O2. SOD1 inhibition increases reactive oxygen species levels and results in increased DNA double strand breaks and carcinogenesis [22].
SOD2 is a key antioxidant, and it is localized in mitochondria and is found to act as potential tumor suppressor gene in carcinogenesis [41]. SOD2 has also showed differential expression in different cancers such as cervical [58], lung [59], brain [60], colon [61], breast [62], gastric [63] and salivary cancer [64]. This overexpression has been found linked to metastasis, poor prognosis and lower survival. In another study SOD2 expression has been found to be downregulated in cancerous cells as compared to control cells [24]. In present study SOD2 has found to be downregulated, one of previous study has demonstrating the similar findings as it has been reported that SOD2 showed downregulated expression in breast, esophageal and pancreatic cancers [65].
OGG1-2α is DNA repair gene and low expression of DNA repair gene results in reduced DNA repair activity resulting in more oxidative imbalance [66]. In previous studies OGG1-2α has been found to be down regulated in carcinogenic cells compared to controls suggesting the systems of excision of bases operate without any disability [67]. It has been found downregulated in colorectal cancer [26] and gastric cancer, which might be due to genetic heterogeneity in OGG1-2α gene because of environmental, genetic, and epigenetic factors [68].
Overexpression of OGG1-2α has been found in one study in lung cancer which is because of the polymorphism Ser326Cys. It has been found that variation of Cys326/Cys326 genotype has high risk of lung cancer than wild type allele carriers, statistically significant interaction was found between genotype and mRNA [69]. In a recent leukemia study, OGG12 has been found downregulated as it is key factor of cancer cells that DNA repair systems are deregulated hence decreased expression of the gene [70]. In present study, expression of OGG12 is found to be downregulated in glioma tissues compared to controls thus, strongly in accordance with previously published results [71].
PARP1 senses the nicks in single stranded and double stranded DNA breaks and seals them caused by oxidative stress. It is found to be hyperactive in oxidative stress and this hyperactivation also leads to cell death [72]. The stressful conditions like inflammation excitotoxicity, ischemia, and oxidative stress cause over activation of PARP1 [73]. In previous studies it has been found to be overexpressed in various tumors like breast, ovary, skin, brain and lungs and this upregulation is linked to the enhanced anti-apoptotic property of tumors [74]. It has also found overexpressed in NSCLC [75], prostate [76], colorectal cancer [77] and gliomas [74]. Overexpression of PARP1 is linked to poor survival overall in cancer [74]. In current study the expression of PARP1 was also found over expressed. PARP1 is found to be linked with the mechanisms relating to proapoptotic mechanisms [76] and overexpression might be due to defective cleavage of PARP1 and reduced apoptosis hence long survival of cancer cells [33].
HIF1α is main regulator of hypoxia, cell cycle, metastasis, angiogenesis, metabolism and proliferation [22,78]. Over expression of HIF1α has been observed in different cancers such as gastric [79] pancreatic cancer [80], NSCLC [81], thyroid cancer [82] and leukemia [83]. In current study HIF1α was found to be upregulated in glioma as compared to the controls supporting the previous results.  has reported that upregulation of HIF1α was found to be associated with activation of other oncogenic pathways like anti-tumor immunosuppression, cell cycle, EMT etc. This association has showed tumor promoter role of HIF1α [22]. HIF1α is stabilized with high ROS levels and provides advantage to tumor cells in division [78].
GDH plays important role in glutamine metabolism and its role in tumor cells is of great interest. GDH plays key role in glutathione which is important for maintaining redox balance in cell [84]. In this study GDH is found to be downregulated in glioma compared to healthy control samples. Deregulation of GDH has been observed in several cancers like thyroid cancer [85], head and neck cancer [86], breast cancer [87], lung cancer [21] and gliomas [88]. It has been found downregulated in lung cancer patients [89]. Expression level of mitochondrial sirtuins was correlated with selected related genes and significant correlation was observed between mitochondrial sirtuins and selected genes. This correlation results showed the combine involvement of selected gene in increased risk of glioma. Diagnostic and prognostic values of selected genes were also measured in present study. ROC curve analysis was performed and >80 percent diagnostic sensitivity was measured in case of SIRT3 and SIRT5 genes in glioma patients. In case of prognostic values of selected genes Kaplan Meier analysis was performed and significant deregulated expression of mitochondrial sirtuins was found linked with the decreased survival rate in glioma patients. Tan et al., (2020) has also reported that deregulation of mitochondrial sirtuins has found associated with decrease survival and increased hazard ratio of lower grade glioma [90]. Previous study has reported the downregulation of SIRT5 gene in glioblastoma and this deregulation was found associated with poor prognosis [91]. Prognostic values of mitochondrial sirtuins have been reported in different cancers such as breast cancer, colon cancer and non-small cell lung carcinoma [92]. Fu et al., (2020) has reported the prognostic value of mitochondrial situins in prostate cancer [93].
In present study DNA damage was measured by single cell gel electrophoresis and significant high number of comets were observed in glioma patients compared to the controls.
Along with this, some other parameters like %DNA in tail, olive tail moment and tail length were also analyzed, and they were found significantly high in glioma samples compared to healthy samples. DNA damage is any physical or chemical change in DNA in cells, in its result interpretation and transmission of genetic information is affected. The damage can be done by multiple factors like chemicals, radiations free radicles and topological changes which ultimately all result in different forms of damage. ROS is well recognized DNA damage mediator [94]. In present study, oxidative stress was also assessed by measuring the antioxidant level and significant increased oxidative stress was observed. ROS also induces DNA damage by oxidizing nucleoside bases (8-oxo guanine) which can lead to G-A and G-T transversions if unrepaired. ROS accumulation also results in mitochondrial DNA lesions, strand breaks and degradation of mitochondrial DNA [95].
Metabolic assays like ATP, NAD+ and glutathione level determination were also performed. In gliomas metabolic pathways like glycolysis, lipid and amino acid metabolism are found to be abnormally regulated. As ATP is the core of these pathways it has inevitably significant impact on glioma behaviors [96]. ATP is found to be at low levels in extracellular fluids in healthy tissues. While in case of some diseases including cancer, ATP level is increased [97], like in gastric cancer [98]. In our study level of ATP was also found high compared to the healthy controls.
Nicotinamide adenine dinucleotide (NAD) is a co-enzyme that actively involved in regulation of metabolic pathway. These pathways are serine biosynthesis, glycolysis and tricarboxylic acid (TCA) cycle [99,100]. As NAD is restored so frequently and act as fuel for tumor cell, promote the uncontrolled cell division, proliferation, and survival. Elevated levels of NAD enhance glycolysis by the GAPDH and LDH, as they require NAD as coenzyme. NAD also serves as substrate for sirtuins and PARP hence mediate deacetylation and poly-ADP-ribosylation [101]. Intracellular NAD in mammals is controlled by two enzymes nicotinamide phosphoribosyltranserfase (NAMPT) and nicotineamide mononucleotide adenyltransferase (Nmnat) [100]. In mammals Nmnat have three isozymes and they have different subcellular locations. NMNAT1 in nucleus, Nmnat2 in golgi apparatus, NMNAT3 in mitochondria and NAMPT is located in cytoplasm [102,103]. In mitochondria, NAD utilization is in TCA cycle, oxidative phosphorylation, and Fatty acid oxidation [104]. In our study NAMPT, NMNAT1 and NMNAT3 were found significantly upregulated in glioma samples compared to the controls. Song et al., (2013) has reported that NMNAT1 is found located on chromosomal region which gets deleted frequently in cancers such as in lung cancer, ultimately resulting in advantage to tumor development [105]. Limited number of studies have been reported for NMNAT3. Anderson et al., (2017) has reported increased expression of NMNAT3, NAD level and enhanced energy level in mouse model [106]. NAMPT has been reported upregulated in different cancers like, colorectal cancer [107] prostate, breast and gastric cancers and this dysregulation is linked to promote tumor glycolysis, metastasis, invasion, proliferation, survival, and chemotherapy resistance [108].

Conclusion
The present study demonstrated the expression variation of selected genes in glioma patients. The deregulation of these genes along with other metabolism deregulation resulted in increased oxidative stress hence resulting in unrepairable DNA damage and reduced survival in glioma patients. This study also provides potent insights into use of mitochondrial sirtuins and related genes as diagnostic and prognostic biomarkers. Several limitations are needed to be considered in present study such as study should incorporate oxygen consumption rate of glioma patients using seahorse analysis for better understanding of role of mitochondrial abnormalities in glioma patients. Our study size is small, further validation studies with large sample size should be done to illuminate the mechanistic role of selected gene in cancerogenesis of different region including glioma.